Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins.

In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2. It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism. In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation. To test this model, we analyzed five temperature-sensitive alleles of the yeast KAR2 gene. When cells carrying a mutation in the Kar2 substrate-binding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress. Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway. Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate. We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2.

[1]  Xi Chen,et al.  ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. , 2002, Developmental cell.

[2]  Randal J. Kaufman,et al.  The Protein Kinase/Endoribonuclease IRE1α That Signals the Unfolded Protein Response Has a Luminal N-terminal Ligand-independent Dimerization Domain* , 2002, The Journal of Biological Chemistry.

[3]  Hiderou Yoshida,et al.  IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. , 2002, Genes & development.

[4]  Stevan R. Hubbard,et al.  IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA , 2002, Nature.

[5]  K. Mori,et al.  XBP1 mRNA Is Induced by ATF6 and Spliced by IRE1 in Response to ER Stress to Produce a Highly Active Transcription Factor , 2001, Cell.

[6]  Randal J. Kaufman,et al.  Complementary Signaling Pathways Regulate the Unfolded Protein Response and Are Required for C. elegans Development , 2001, Cell.

[7]  P. Walter,et al.  Block of HAC1 mRNA Translation by Long-Range Base Pairing Is Released by Cytoplasmic Splicing upon Induction of the Unfolded Protein Response , 2001, Cell.

[8]  J. Brodsky,et al.  Molecular Chaperones in the Yeast Endoplasmic Reticulum Maintain the Solubility of Proteins for Retrotranslocation and Degradation , 2001, The Journal of cell biology.

[9]  T. Iwawaki,et al.  Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress , 2001, Nature Cell Biology.

[10]  K. Okamura,et al.  Dissociation of Kar2p/BiP from an ER sensory molecule, Ire1p, triggers the unfolded protein response in yeast. , 2000, Biochemical and biophysical research communications.

[11]  R. Kaufman,et al.  Ligand-independent Dimerization Activates the Stress Response Kinases IRE1 and PERK in the Lumen of the Endoplasmic Reticulum* , 2000, The Journal of Biological Chemistry.

[12]  Anne Bertolotti,et al.  Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response , 2000, Nature Cell Biology.

[13]  K. Mori,et al.  mRNA splicing-mediated C-terminal replacement of transcription factor Hac1p is required for efficient activation of the unfolded protein response. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[14]  A. Ohta,et al.  Unfolded protein response-induced BiP/Kar2p production protects cell growth against accumulation of misfolded protein aggregates in the yeast endoplasmic reticulum. , 1999, European journal of cell biology.

[15]  T. Rapoport,et al.  BiP Acts as a Molecular Ratchet during Posttranslational Transport of Prepro-α Factor across the ER Membrane , 1999, Cell.

[16]  J. Brodsky,et al.  The Requirement for Molecular Chaperones during Endoplasmic Reticulum-associated Protein Degradation Demonstrates That Protein Export and Import Are Mechanistically Distinct* , 1999, The Journal of Biological Chemistry.

[17]  D. Ron,et al.  Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase , 1999, Nature.

[18]  Masahiko Kuroda,et al.  Cloning of mammalian Ire1 reveals diversity in the ER stress responses , 1998, The EMBO journal.

[19]  R. Kaufman,et al.  A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells. , 1998, Genes & development.

[20]  L. Hendershot,et al.  BiP Maintains the Permeability Barrier of the ER Membrane by Sealing the Lumenal End of the Translocon Pore before and Early in Translocation , 1998, Cell.

[21]  Bernd Bukau,et al.  The Hsp70 and Hsp60 Chaperone Machines , 1998, Cell.

[22]  Peter Walter,et al.  The Transmembrane Kinase Ire1p Is a Site-Specific Endonuclease That Initiates mRNA Splicing in the Unfolded Protein Response , 1997, Cell.

[23]  R. Fukuda,et al.  Accumulation of Misfolded Protein Aggregates Leads to the Formation of Russell Body‐like Dilated Endoplasmic Reticulum in Yeast , 1997, Yeast.

[24]  R. Plemper,et al.  Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation , 1997, Nature.

[25]  K. Mori,et al.  Saccharomyces cerevisiae KAR2 (BiP) gene expression is induced by loss of cytosolic HSP70/Ssa1p through a heat shock element-mediated pathway. , 1997, Journal of biochemistry.

[26]  P. Walter,et al.  A Novel Mechanism for Regulating Activity of a Transcription Factor That Controls the Unfolded Protein Response , 1996, Cell.

[27]  R. Kaufman,et al.  The Unfolded Protein Response Pathway in Saccharomyces cerevisiae , 1996, The Journal of Biological Chemistry.

[28]  P. Walter,et al.  Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. , 1996, The EMBO journal.

[29]  R. Schekman,et al.  BiP and Sec63p are required for both co- and posttranslational protein translocation into the yeast endoplasmic reticulum. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Simons,et al.  BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast , 1995, The Journal of cell biology.

[31]  M. Aebi,et al.  The genetic interaction of kar2 and wbp1 mutations. Distinct functions of binding protein BiP and N-linked glycosylation in the processing pathway of secreted proteins in Saccharomyces cerevisiae. , 1994, European journal of biochemistry.

[32]  R. Fukuda,et al.  The prosequence of Rhizopus niveus aspartic proteinase-I supports correct folding and secretion of its mature part in Saccharomyces cerevisiae. , 1994, The Journal of biological chemistry.

[33]  H. Okamura,et al.  Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum. , 1993, Molecular biology of the cell.

[34]  J. Sambrook,et al.  A transmembrane protein with a cdc2+ CDC28 -related kinase activity is required for signaling from the ER to the nucleus , 1993, Cell.

[35]  Peter Walter,et al.  Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase , 1993, Cell.

[36]  J. Sambrook,et al.  The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum , 1993, Molecular and cellular biology.

[37]  M. Tokunaga,et al.  Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae. , 1992, The Journal of biological chemistry.

[38]  J. Sambrook,et al.  A 22 bp cis‐acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins. , 1992, The EMBO journal.

[39]  R. Schekman,et al.  Sec61p and BiP directly facilitate polypeptide translocation into the ER , 1992, Cell.

[40]  M. Dante,et al.  Multifunctional yeast high-copy-number shuttle vectors. , 1992, Gene.

[41]  T. Ashikari,et al.  High-level secretion of a Rhizopus niveus aspartic proteinase in Saccharomyces cerevisiae. , 1990, Agricultural and biological chemistry.

[42]  M. Rose,et al.  Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast , 1990, The Journal of cell biology.

[43]  J. Sambrook,et al.  S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP , 1989, Cell.

[44]  M. Rose,et al.  KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene , 1989, Cell.

[45]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[46]  K. Kohno,et al.  Inhibition of Biosynthesis of Polyisoprenol Sugars in Chick Embryo Microsomes by Tunicamycin , 1975 .

[47]  J. Strathern,et al.  Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual , 2005 .

[48]  J. Polaina,et al.  Genes involved in the control of nuclear fusion during the sexual cycle of Saccharomyces cerevisiae , 2004, Molecular and General Genetics MGG.

[49]  G. Fink,et al.  Methods in yeast genetics , 1979 .